Kaplan H, Hefford M A, Chan A M, Oda G
Biochem J. 1984 Jan 1;217(1):135-43. doi: 10.1042/bj2170135.
A modification to the competitive labelling procedure of Duggleby and Kaplan [(1975) Biochemistry 14, 5168-5175] was used to study the reactivity of the N-termini, lysine, histidine and tyrosine groups of insulin over the concentration range 1 X 10(-3)-1 X 10(-7)M. Reactions were carried out with acetic anhydride and 1-fluoro-2,4-dinitrobenzene in 0.1 M-KCl at 37 degrees C using Pyrex glass, Tefzel and polystyrene reaction vessels. At high concentrations all groups had either normal or enhanced reactivity but at high dilution the reactivities of all functional groups became negligible. This behaviour is attributed to the adsorption of insulin to the reaction vessels. The histidine residues show a large decrease in reactivity in all reaction vessels in the concentration range 1 X 10(-3)-1 X 10(-5)M where there are no adsorption effects and where the reactivities of all other functional groups are independent of concentration. With polystyrene, where adsorption effects become significant only below 1 X 10(-6)M, the reactivity of the phenylalanine N-terminus also shows a decrease in reactivity between 1 X 10(-5) and 1 X 10(-6)M. In 1 M-KCl insulin does not absorb to Pyrex glass and under these conditions the histidine reactivity is concentration-dependent from 1 X 10(-3) to 5 X 10(-6)M and the B1 phenylalanine alpha-amino and the B29 lysine epsilon-amino reactivities from 5 X 10(-6) to 1 X 10(-7)M, whereas the reactivities of all other groups are constant. These alterations in reactivity on dilution are attributed to disruption of dimer-dimer interactions for histidine and to monomer-monomer interactions for the phenylalanine and lysine amino groups. It is concluded that the monomeric unit of insulin has essentially the same conformation in its free and associated states.
采用对Duggleby和Kaplan[(1975)《生物化学》14, 5168 - 5175]竞争标记程序的一种改进方法，研究胰岛素的N端、赖氨酸、组氨酸和酪氨酸基团在1×10⁻³ - 1×10⁻⁷M浓度范围内的反应活性。反应在37℃下于0.1M - KCl中，使用派热克斯玻璃、特氟龙和聚苯乙烯反应容器，分别与乙酸酐和1 - 氟 - 2,4 - 二硝基苯进行。在高浓度时，所有基团的反应活性正常或增强，但在高稀释度下，所有官能团的反应活性变得可忽略不计。这种行为归因于胰岛素吸附到反应容器上。在1×10⁻³ - 1×10⁻⁵M浓度范围内，在没有吸附效应且所有其他官能团的反应活性与浓度无关的情况下，组氨酸残基在所有反应容器中的反应活性大幅下降。对于聚苯乙烯，吸附效应仅在低于1×10⁻⁶M时才显著，苯丙氨酸N端的反应活性在1×10⁻⁵至1×10⁻⁶M之间也呈现下降。在1M - KCl中，胰岛素不吸附到派热克斯玻璃上，在此条件下，组氨酸的反应活性在1×10⁻³至5×10⁻⁶M范围内与浓度相关，B1苯丙氨酸α - 氨基和B29赖氨酸ε - 氨基的反应活性在5×10⁻⁶至1×10⁻⁷M范围内与浓度相关，而所有其他基团的反应活性保持恒定。稀释时反应活性的这些变化归因于组氨酸的二聚体 - 二聚体相互作用的破坏以及苯丙氨酸和赖氨酸氨基的单体 - 单体相互作用的破坏。得出的结论是，胰岛素的单体单元在其游离和缔合状态下基本具有相同的构象。
